Extended Data Fig. 8: IEG bipartite chromatin is necessary to prevent precocious activity-dependent neuronal maturation.

a, Short time (two days) E12.5 ex vivo-cultured Drg11^tdTomato/+ hindbrains. After over-night (o/n) treatment with a cocktail of neuronal activity blockers (TDN cocktail = TTX + D-AP5 + NBQX, inhibitors of sodium channel, NMDAR and AMPAR), cultured neurons were treated by 55 mM KCl for 1 hour. Drg11-positive immature trigeminal neurons were FACS-isolated for ATAC-seq analysis. Violin plots visualize log2 fold changes of enhancer chromatin accessibilities in 1 hour KCl-treated neurons as compared to non-treated control neurons. Increased accessibility is selectively detected in KCl-treated neurons at activity-dependent Fos-binding enhancers that normally become open only at P4 (green, n = 85 enhancers) (purple, all non-Fos-binding enhancers that gain accessibilities only at P4, n = 3882 enhancers). Plots extend from the data minima to the maxima with the white dot indicating median, the box showing the interquartile range and whiskers extending to the most extreme data point within 1.5X the interquartile range from the box. P value is from a two-sided Wilcoxon’s test. b, Scatterplots comparing enhancer accessibilities (ATAC) in E14.5 Ezh2 heterozygous control (ctrl) and homozygous mutant (Ezh2cKO^HB-RFP) hindbrain cells. All the barrelette enhancers in Drg11^{vPrV-ZsGreen/+} barrelette neurons (left), non-Fos-binding enhancers that gain accessibilities at P4 as compared with E14.5 in Drg11^{vPrV-ZsGreen/+} barrelette neurons (n = 3882 enhancers, middle, purple), neuronal activity-dependent Fos-binding enhancers that gain accessibilities at P4 as compared with E14.5 Drg11^{vPrV-ZsGreen/+} barrelette neurons (n = 85 enhancers, left, green) are shown (Methods). 85 activity-dependent Fos-binding enhancers show precocious opening upon H3K27me3 removal at E14.5. Also see Fig. 5d.